Anticancer Potential of Ficus religiosa (Linn) Against Human Breast Cancer Cell Line MDA-MB-231 and its Phytoconstituents by HR-LCMS

Background: Ficus religiosa is widely present in India especially in northern region and has been traditionally used for various indications. We investigated the anticancer potential of leaf extract against human breast cancer cell line MDA-MB-231. The anticancer potential was further supported by identifying potential phytoconstituents of the extract by HR-LCMS analysis. Methods: Amongst the collected fractions, subfraction of chloroform was selected for anticancer screening in parallel for identication of phytochemicals by using HR-LCMS. Extract showed positive results in a battery of two tests employed in the current study by using MDA-MB231 breast cancer cell lines. In trypan blue exclusion dye test, 50% of cell killing was observed at 110 µg/ML. In vitro anticancer activity by using SRB assay showed dose dependent growth inhibition at 62.5, 125, 250 and 500 µg/mL concentrations. The parameters LC 50 , GI and TGI 50 found from the assay were 100, 200, 300 µg/mL respectively. Assay parameter In case of HR-LCMS analysis several active phytochemicals were identied in the extract. Results: F. religiosa leaf extract showed promising anticancer activity against human breast cancer cell MDA-MB-231. Conclusion: F. religiosa leaf extract has promising anticancer potential against human breast cancer cells MDA-MB-231 and numerous phytochemicals further warrants evaluating it for multi-therapeutic potential. of direct killing effect as well as disruption of cellular processes of MDA-MB-231 were successfully established in the experiment for the extract of F. religiosa. Strategy of establishing cell killing concentration by Trypan Blue assay made possible to investigate anti-cancer effect of the extract by operating lower than cytotoxic concentrations in SRB assay. Extract demonstrated 25% cytotoxicity at the concentration of 1000 µg/mL in Trypan Blue exclusion assay. Secondly, the extract showed biologically signicant disruptions of cellular functions in human breast cancer cell line MDA-MB-231 at the concentrations of 100 and 200 µg/mL. HR-LCMS analysis data showed presence of number of active compounds which are having various medicinal properties. The identication and analysis of chemically active components in F. religiosa leaves extract could be promising remedy in the future to treat human breast cancer particularly caused due to MDA-MB0-231 cells. The work further warrants in-depth chemical identication and fractionation of other unknown anticancer compounds if any present in extract with their in-vitro, and in-vivo anticancer evaluations.


Background
Nowadays, cancer is considered as one of the most dominant and progressive diseases in the world due to which morbidity and mortality is considered to be very high in records. The disease, not only deteriorate quality of life but also putting high economic burden globally. Treatment and prevention of this disease is necessary by inventing new treatment strategies, economic models, and innovative drugs. Herbal medicines block critical biochemical pathways converting normal cells to cancer cells and check the progress of the disease [1]. Cancer disease starts with the deformation of a natural cell caused by genetic mutations in DNA [2]. Cancer is an important health problem in developing and developed countries.
Cancer is the second leading causes of death worldwide (WHO). The most important problem in cancer treatment is destroying cancerous cells in parallel by protecting body's own normal cells. In order to bring anticancer medicines from natural resources like plants, cytotoxicity testing of compounds and safety screening the raw extracts of plants are essential [3]. Medicinal herbs are valuable assets in making anticancer strategies due to having multiple chemical compounds, secondary metabolites of potential to ght against the disease [4]. Plants produce a wide range of chemical compounds called secondary metabolites; they have no direct role in the growth of plant. Alkaloids, terpenoids, avonoids, pigments, and tannins are important constituents of these compounds. Secondary metabolites exhibit biological effects like anti-in ammatory, anticancer and contraceptive etc [5]. Many studies reported that treatment of cancer with herbal products stand as one of the safe treatments because of no side effects on body's healthy cells [1].
Ficus religiosa (L.) is a huge evergreen tree found throughout India. It is wild as well as cultivated. Commonly known as 'Peepal tree' is a large widely branched tree with leathery, heart shaped, long tipped leaves on long slender petioles and purple fruits growing in pairs [6]. Phytochemical investigation of Ficus religiosa (L.) leaves showed campestrol, stigmasterol, isofucosterol, α-amyrin, lupeol, tannic acid, arginine, serine, aspartic acid, glycine, threonine, alanine, proline, tryptophan, tryosine, methionine, valine, isoleucine, leucine, n-nonacosane, n-hentricontanen, hexa-cosanol and n-octacosan [7]. The extract also demonstrated good anti-i ammatory and wound healing potential in experimental burn model [8,9]. Traditionally used in breast cancer cell lines, induces apoptosis [10]. In Asian countries, F. religiosa (L.) bark is used for treating various diseases like cervical cancer, epilepsy and in ammation [11]. F. religiosa (L.) barks, leaves and fruits are used in various treatments, such as diabetes, skin diseases, ulcers, dysentry, diarrohoea, stomachache, anti-in ammatory, antioxidant and anticancer agent [12]. Traditional herbal practice in the cancer is passed from one generation to the other through oral tradition. Ficus religiosa (L.) leaves and fruits are used for treatment of cancer in Chhattisgarh (India) [13].
Some species of the genus Ficus shows anticancer effects and have been evaluated in vitro by employing different cancer cell lines. Ficus carica leaves and fruits have been studied to de ne anticancer effects on liver cell line Hu7it. The extracts showed effect but at very high concentrations [14]. Leaf extract of Ficus carica decreased proliferation of MDA-MB-231 and induced apoptotic events [15]. Crude extracts of leaves, g and stem bark from Ficus exasperata demonstrated good activity against ovarian cancer cells A2780 in vitro [16]. MDA-MB-231 is breast cancer cell line displays invasiveness by mediating the proteolytic degradation of the extracellular matrix (ECM), including basement membrane and several mechanical barriers to the ECM, through the increased expression of matrix metallo proteinases. It is perfect model for screening of chemotherapeutic agents because it is highly aggressive and differentiated triple-negative breast cancer cell line. It lacks oestrogen receptor and progesterone receptor expression, as well as human epidermal growth factor receptor 2 ampli cation [17,18].

Material And Method
Chemicals, reagents and equipment All the chemicals used in the experiment were of high quality and analytical grade. The chemicals procured for the experiment were methanol, ethyl acetate, hexane, chloroform, ethanol, acetone, 0.4% Trypan Blue dye solution (Sigma), Sulforhodamine B (SRB) solution, DMSO (Dimethyl sulphoxide), microscope (Carl Zeiss) and haemocytometer. Human breast cancer cells were maintained and all in vitro experiments were conducted at was conducted at ACTREC, Mumbai.

Plant material collection and preparation of powder
The mature leaves of Ficus religiosa (L.) were collected from the campus of Dr. Babasaheb Ambedkar Marathwada University, Aurangabad Maharashtra, India in summer 2017 herbarium deposited in Department of Botany, Accession no. 0710. This plant validated taxonomically Sp. Pl. 1059 1753. The details are also found in (IPNI Life Sciences Identi er (LSID) urn:lsid:ipni.org:names:853563-1) Leaves were thoroughly washed with distilled water and completely dried at room temperature. Dried leaves were crushed to make ne powder by using mixer grinder.
Extraction and fractionation of F. religiosa by column chromatography Fine dried powder (250 gm) was sequentially soaked in non-polar to polar solvent as mentioned above in n-hexane, chloroform, ethyl acetate and methanol (1:10 w/v) for preliminary fractionation. Subsequently, next fractionation was carried out at room temperature (around 30 °C) for 12 hours with intermittent shaking of extraction ask and nally ltered through with whatman lter paper (Whatman no. 41).
Resulting extracts were n-hexane, chloroform, ethyl acetate and methanol. Chloroform extract was evaporated to dryness and the resulting weight of extract was 3.2 gm (sticky and brown colour). This extract was subjected to column chromatography using silica gel of 120-160 mesh. Hexane and ethyl acetate were used for elution in different ratio  Table 2). The percentage of viable cells was calculated by dividing the number of viable cells by the number of total cells and multiplying by 100 or % viable cells = [1-(Number of blue cells ÷ Number of total cells)] × 100. Cytotoxic concentration (CC 50 ) was determined by using % viable cells data obtained at various concentrations by feeding in statistical analysis [19].
In vitro anticancer activity of F. religiosa extract in MDA-MB-231 cell line Exponentially growing cells were con rmed and prepared cell suspension in DMEM at the densities of 3 × 10 3 in 100 µL. In a 96 well plate, 100 µL of cell suspension was seeded in each well and incubated at 37 0 C, 5% CO 2 and 95% humidity for 24 hours. Next day before addition of test substance monolayer of the cells was washed prior to addition of experimental substance. 5 mg of dried leaves extract was weighed in microfuge tube and about 500 uL of DMSO (Dimethyl sulphoxide) was added and extract was completely solubilised by vortex mixing. Final volume was adjusted to 5 mL by adding sterile water for injection to become nal strength of 1 mg/mL (1000 µg/mL) of dried sample extract. The strength of 1000 µg/mL was further diluted to prepare the concentrations of 25, 50, 100 and 200 µg/mL by adding sterile water for injection. Aliquots of 10 µL of all 4 dilutions of the samples were added to the wells already containing 90 µL of medium, resulting in the required nal concentrations in an ascending order of 25, 50, 100 and 200 µg/mL exposed to the breast cancer cell line (Table 3). Standard drug Adriamycin was kept as positive control along with the test substance at the concentrations of 10, 20, 40 and 80 µg/mL in the same pattern ( Fig. 1). After addition of substances, plates were incubated at standard conditions for 48 hours and assay was terminated by the addition of cold TCA. Cells were xed in situ by the gentle addition of 50 µl of cold 30% (w/v) TCA ( nal concentration, 10% TCA) and incubated for 60 minutes at 4 °C. The supernatant was discarded; the plates were washed ve times with tap water and air dried. Sulforhodamine B (SRB) solution (50 µl) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, and plates were incubated for 20 minutes at room temperature. After staining, unbound dye was recovered and the residual dye was removed by washing ve times with 1% acetic acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was read on plate reader at a wavelength of 540 nm with 690 nm reference wavelength. During the treatment, standard environmental conditions were maintained till end of the treatment. There were three replicates of the experiment were performed at all the selected concentrations from sample and standard drug and average data was calculated and presented. This experiment was conducted at ACTREC Mumbai, India.

Statistical analysis
For cytotoxicity experiment, data of three independent experiments were evaluated and presented as mean and ± SD. The CC 50 in µg/mL of the extract was determined from three run of the experiment and presented as mean. For SRB assay, percent growth was calculated on the basis for test wells relative to control wells. Percent cell growth of extract was expressed as the ratio of average absorbance of the test well to the average absorbance of the control wells x100. % Cell growth = Average absorbance of extract/Average absorbance in controlx100. Using six absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of the extract at the four concentration levels (Ti), the percentage growth was calculated at each of the test sample concentration levels. Percentage growth inhibition was calculated as [Ti/C] x 100% [20,21]. Using Finney's method of probit analysis, CC 50 and 50% affected growth was determined from average values of 3 experiments [22].
Identi cation of phytoconstituents from leaves extract of F. religiosa by HR-LCMS Metabolites were identi ed from subfraction of chloroform extract was carried out at Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology, Bombay. Samples were analyzed on an LC-ESI-Q-TOF-MS (Agilent Technologies G6550A) system equipped with a G4220B pump, G4226A auto sampler and G1316C, and a diode array detector. The elution solvent consisted of a gradient system of 0.1% formic acid in water (A) and acetonitrile (B) at a ow rate of 0.2 ml/min. The gradient system started with 95:5% A: B reaching 5:95% A:B in 30 min, then back to the initial composition 15 min, which was held at the same composition for 2 min. The MS analysis was carried out using an ESI-positive ionization mode. MS source conditions were as follows: capillary voltage 3,500 V; gas temperature, 250 °C; drying gas ow, 13 L/min; sheath gas temperature, 300 °C; sheath gas ow, 11; nebulizing gas pressure, 35 (psig); fragmentor, 175 V; skimmer, 65 V; Octopole RF Peak, 750 V; and mass range m/z 50-1,000. The resolution was 40,000 FWHM. The Metlin database was used for structure con rmation [23].

Result
In-vitro cytotoxicity Cytotoxicity was determined by counting number of dead cells present among the total counted cells on Neubauer chamber and expressed in % dead cells at respective concentrations. The percent dead cells found were 5%, 12%, 18% and 25% at the concentrations of 125, 250, 500 and 1000 µg/mL respectively after incubation with the extract. Extract found mild cytotoxic at highest selected concentration of 1000 µg/mL. The calculated CC 50 (Table 4). It was observed that leaves extract at the concentrations of 100 and 200 µg/mL caused signi cant retardation of cell growth in 48 hours of incubation as compared to growth of untreated cells. In Adriamycin treated arm, there was no growth observed in all the selected concentrations, the % growth values were found negative due to high rate of cytotoxicity without growth of the cells occurred up to 48 hours of incubation. Hence, results of Adriamycin validate the experimental set-up and its anticancer potential on MDA-MB-31 cell lines. From above parameters, lethal concentration and total inhibition of the cells were calculated. The concentration of 180 µg/mL of leaves extract caused 50% lethality of the cells (LC 50 ), whereas total growth inhibition of the cells occurred at the concentration of 120 µg/mL and the 50% inhibition of the cell growth (GI 50 ) was found to be at the concentration of 70 µg/mL (Table 5).
Average % control affected growth was calculated as 100-% control growth values i.e. 0.6, 4.1, 26.9 and 78.5% at the concentrations of 25, 50, 100 and 200 µg/mL, respectively. On the basis of these values, 50% affected growth was found at 130.503 µg/mL (with the con dence interval of (51.489 to 330.774 µg/mL). Breast cancer cells in 48 hours of treatment with leaves extract sample showed reduced density and morphology of cells as compared to control. Overall, the results of the study indicated that the growth inhibition and killing potential of the extract occurred at higher concentrations (Fig. 2, 3.1 and   3.2). Standard drug, Adriamycin showed satisfactory results at selected concentration, which con rms the validity of experiment. Due to absent of growth proteins of the cells from the lowest concentration of 10 µg/mL of Adriamycin, TGI and GI 50 parameters were found to be < 10 µg/mL and % control growth values were found negative. Cell growth after 48 hours of Adriamycin treatment showed reduced density (Fig. 2, 3.1 and 3.2).

F. religiosa leaves extraction and identi cation of phytochemicals
Fine dried powder was sequentially soaked in non-polar to polar solvents. Among all extractions the chloroform extract was further fractionated by column chromatography using n-hexane and ethyl acetate. In this study, total 100 compounds, including Convallatoxin, Umbelliferone, Embelin, Dihydrocoumarin, Fendiline, Hydroxyibuprofen (Table 1) were detected in the chloroform extract of F. religiosa (L.) leaves as active compounds by HR-LCMS. The above work was done at IIT, Mumbai, India.

Convallatoxin
Convallatoxin is natural glycoside found in many organisms, including plants. This substance is reported as inhibitor of cancer cell growth [24]. It is also reported as effective anti-proliferative agent against some cancer types [25,26]. In another study, it is demonstrated good activity against colorectal type of cancer [27]. It also exerts good cytotoxic effects against ER + breast cancer cell lines, MCF-7 cells [25]. (Fig. 4A).

Umbelliferone
Umbelliferone is a phenylpropanoid and it is coumarin and widely spread in plants28. Umbelliferone has found as antioxidant in the literature [29]. It is used as a sunscreen agent and as optical brightener in textiles [30]. It has anti-in ammatory [31], anti-diabetic [32] and anti-tumour activity [33]. Umbelliferone also have glycation inhibitory activity [34], angiotensin converting enzyme inhibitory activity [35].
Antinociceptive [36], antimicrobial [37], neuroprotective [38], hepatoprotective [39], anti-in ammatory and pro apoptotic effects in colon carcinogenesis [40]. (Fig. 4B) Embelin Embelin is naturally occurring parabenzoquinone isolated from plant sources. It inhibits cell growth, induces apoptosis, and activates caspase-9 in prostate cancer cells with high levels of inhibitor of apoptosis family of proteins called XIAP, but has a minimal effect on normal prostate epithelial and broblast cells with low levels of XIAP [41]. It is used for treating various ailments like chronic in ammatory disorders, heart and urinary conditions, snake and insect bites, and tumour [42]. Embelin would be useful as an adjunct therapy for cerebral stroke and as a potent neuroprotective agent [43]. It possesses all the characteristics of a compound which can cross the blood-brain barrier and elicit an effect on the CNS [44]. Embelin is reported for its CNS effect by diverse mechanisms. (Fig. 4C).

Dihydrocoumarin
Coumarins are natural and synthetically active components used for various pharmacological actions. It is widely useful in various types of medicinal treatments, such as cancer, in ammation, infection, and enzyme inhibition [45]. Dihydrocoumarin (DHC) is an inhibitor of the Sirtuin family of NAD + -dependent deacetylases [46] 4-arylcoumarins and 3, 4-dihydro-4-arylcoumarins were synthesized and tested for antioxidant activity, antitumor activity [47]. 3, 4-Dihydrocoumarins, considered to be valuable building blocks, have attracted considerable attention due to their various biological activities [48] such as aldose reductase inhibition [49], protein kinases [50] antiherpetic and avouring agent to a diverse set of foods for example-soft drinks, yogurt, mu ns) [51]. In addition, the 3,4dihydrocoumarin scaffold has been discovered in a number of important natural compounds as exempli ed by Calomelanol A-C, E-J [52]. (Fig. 4D).

Fendiline
Fendiline inhibits cancer cell proliferation, speci cally cells expressing mutant K-Ras, by altering K-Ras cellular distribution and downstream signalling [53]. It inhibits the maintenance and expression of methamphetamine [54]. The effects of Fendiline were produced in the absence of aversive or rewarding properties and without signi cant impairment on motivated motor behaviour [55]. It was primarily developed as L-type Ca 2 + channel blocker for cardiac smooth muscles and approved for human use to treat angina, hypertension and cardiac arrhythmias [56]. (Fig. 4E).
Hydroxyibuprofen 2-Hydroxyibuprofen is a metabolite of ibuprofen. Ibuprofen is an anti-in ammatory drug (NSAID) and act as cyclooxygenase-inhibitor [57]. It is also used for symptoms of arthritis, fever, and as an analgesic.
Ibuprofen is known to have an antiplatelet effect as well. (Fig. 4F).

Discussion
Ficus religiosa (L.) plant recently attracted global attention towards its tremendous medicinal properties against various lifestyle diseases and infections. Since cancer is the second leading disease to cause mortality and morbidity worldwide, hence there is urgent need to bring novel and effective treatment in broad sense. Firstly, cytotoxic concentrations were determined for the extract to operate the concentrations for anticancer properties by using SRB assay. De ning 25% cytotoxicity at the highest concentration of 1000 µg/mL by Trypan Blue method further became easy to select lower concentrations for next SRB assay for investigating anticancer properties. Selection of two in vitro assays in the current work could differentiate the frank cellular toxicity and interruption of cell functions. Due to selection of Trypan Blue assay, it was possible to know the killing properties of the extract and helped in establishing the direct killing concentration [58]. On the other hand, selection of SRB assay in the work successfully determined that at what concentrations, the extract caused disturbances in the cellular functions of the cancer cells mainly during growth and proliferation stages of the cells [59]. Speci city of SRB dye towards protein ultimately determines cellular proliferation, growth of the cells. The Decrease in absorbance of the dye in sample extract directly impacted cell number, growth of individual cell or cell cycle. HR-LCMS analysis of extract showed presence of several pharmacologically active compounds with documented anticancer potential. Mode of action of these compounds could be different but in broad sense of available information it has been found that the effect is through inducing oxidative stress which ultimately causing death of the cancer cells. Selection of highly invasive, commonly causing breast cancer cells, MDA-MB-231gives an advantage of studying the extract for future development of anticancer therapy [60]. Recently it has been investigated that F. religiosa (L.) induced apoptosis in ovarian cancer cell lines Jurkat cancer cells indicates that substance mixture has active chemicals that interferes in anti-apoptotic pathways of cancer cells [61]. Finally, validity of the SRB assay was con rmed by using positive drug Adriamycin at validated concentrations along with the experiment to con rm end point results are as per standard operating conditions of the experiment.   Table 3 Concentrations